Structure - Dynamics Relationships in Proteins: Multi-faceted characterization of structure and its fluctuations by NMR relaxation measurements and molecular dynamics simulations. Protein fluctuations and their relationship to protein structure and function continue to challenge biophysical measurements and simulations. All three aspects of proteins (dynamics, structure and function) are intimately linked. Recently it has become clear that alterations in protein dynamics alone can be used to communicate between distant sites in proteins. The structures and fluctuations that are involved in such communication conduits (and the coupling between them) are not yet well understood. We address several aspects of the structure-dynamics relationship. Aim 1: In a joint NMR experimental -computational refinement approach we seek to improve the representation of ps-ns timescale dynamics in protein structural ensembles. A statistical and an information theory based approach will be employed to evaluate the experimental restraints and the number of local conformers to be used in the structure refinement. The results will be compared to unrestrained molecular dynamics simulations and to order parameters derived from NMR relaxation measurements. The structural ensembles will be useful for ligand and protein docking calculations in drug design. Aim 2: Our study seeks to reveal design principles that allow a coupling between protein loops and the fluctuations of core structures. Again, both solution NMR experimental and computational strategies will be combined for several proteins, including for ubiquitin and for the RhoGTPase binding domain of plexin-B1 (RBD), which has a ubiquitin fold with long loop insertions. The loops as well as the protein core will be manipulated in order to probe possible dynamic coupling between the two. Possible motional coupling across a protein-protein interface will also be examined for the RBD-GTPase complex. Aim 3: The possibility that the global stochastic motion of the protein can affect the local, internal dynamics will be examined using NMR relaxation at different solvent viscosities and long time-scale Langevin/Brownian dynamics simulations. Aim 4: Methods for enhanced sampling of conformational space will be tested and a next generation force field for the molecular dynamics program CHARMM/NAMD will be validated against NMR data. Overall, in this project, several computational and experimental strategies will be brought together in order to provide deep insight into the relationship between protein structures, and internal as well as global protein dynamics. Several of the proteins studied have important roles in oncogenesis and cell metastasis and their further investigation will suggest new avenues for the design of diagnostic or therapeutic agents to combat cancer. PUBLIC HEALTH RELEVANCE: The joint experimental and computational project will provide detailed insight into the interrelationship between protein structure and protein internal and global dynamics. The basic questions addressed are fundamental to the field of protein biophysics and structural biology. The results of this study will help to understand protein function, here specifically of cell signaling proteins. Several of the proteins involved play important roles in cancer development and spreading, and their further investigation will suggest new avenues for the design of diagnostic or therapeutic agents.